Hepatic steatosis aggravates atherosclerosis via small extracellular vesicle-mediated inhibition of cellular cholesterol efflux.

BACKGROUND & AIMS: While it is recognized that non-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease (CVD), how NAFLD affects the development and progression of CVD remains unclear and debatable. Hence, we aimed to determine the role of steatotic hepatocyte-derived small extracellular vesicles (sEVs) in foam cell formation and atherosclerosis progression. METHODS: sEVs from steatotic hepatocytes were isolated and characterized. MicroRNA (miRNA) deep sequencing was utilized to identify functional miRNA in sEVs. Lastly, we conducted a cross-sectional study on patients with NAFLD to validate these findings. RESULTS: Treatment of sEVs from steatotic hepatocytes promoted macrophage-derived foam cell formation and atherosclerosis progression via inhibition of ABCA1-mediated cholesterol efflux. Macrophage-specific deletion of Abca1 in ApoE-/- mice abolished the role of steatotic hepatocyte-derived sEVs in atherosclerosis progression. In addition, hepatocyte-specific deletion of Rab27a, which is the key GTPase regulating sEV release, significantly ameliorated high-fat, high-cholesterol diet-induced atherosclerosis progression in ApoE-/- mice. The miRNA deep sequencing results showed that miR-30a-3p was enriched in sEVs from steatotic hepatocytes. miR-30a-3p directly targeted the 3' untranslated region of ABCA1 to inhibit ABCA1 expression and cholesterol efflux. Treatment with antagomiR-30a-3p significantly attenuated atherosclerosis progression in high-fat, high-cholesterol diet-fed ApoE-/- mice. Moreover, serum sEVs from patients with NAFLD and sEV-miR-30a-3p expression were associated with decreased cholesterol efflux levels in foam cells. CONCLUSION: Steatotic hepatocyte-derived sEVs promote foam cell formation and facilitate atherogenesis via the miR-30a-3p/ABCA1 axis. Reducing sEV secretion by steatotic hepatocytes or targeting miR-30a-3p may be potential therapeutic approaches to slow the progression of NAFLD-driven atherosclerosis. IMPACT AND IMPLICATIONS: The presence of hepatic steatosis is strongly correlated with the risk of cardiovascular disease and cardiovascular events, yet the molecular mechanisms linking steatosis to progression of atherosclerosis are unclear. Herein, we identified small extracellular vesicles from steatotic hepatocytes as a trigger that accelerated the progression of atherosclerosis. Steatotic hepatocyte-derived small extracellular vesicles promoted foam cell formation via the miR-30a-3p/ABCA1 axis. Our findings not only provide mechanistic insight into non-alcoholic fatty liver disease-driven atherosclerosis but also provide potential therapeutic targets for patients with atherosclerosis.

Associations of MAFLD and MAFLD subtypes with the risk of the incident myocardial infarction and stroke.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a condition characterized by liver fat accumulation and metabolic abnormalities. Given the potential impact of MAFLD on patient health, it is important to understand its association with major adverse cardiovascular events (MACE) such as myocardial infarction (MI) and stroke. In the prospective UK Biobank cohort, we sought to elucidate the association of MAFLD and its subtypes with incident MI and stroke. In this study, we analyzed the data of 325,129 participants in the UK Biobank and calculated relative risks for MI and stroke using Cox regression analysis. Among 325,129 participants over a median duration of 12.8 years follow-up, participants with MAFLD were significantly more likely to experience a MI (hazard ratio [HR] = 1.35, 95% confidence interval [CI: 1.29;1.41] P < 0.001) or a stroke (HR = 1.26 [1.18-1.33] P < 0.001) compared to those without MAFLD. In addition, diabetic, overweight with metabolic dysfunction (MD), and lean MAFLD subtypes were significantly associated with an increased risk for MI and stroke, whereas overweight without MD subtype did not appear to be associated with this risk. Our findings also revealed graded associations between liver fibrosis scores and risk of MI and stroke in MAFLD patients. However, only diabetic, and overweight patients with MD subtypes exhibited graded associations between liver fibrosis score and the risk of MI and stroke among the MAFLD subtypes. Furthermore, the risk alleles traits of fatty liver did not enhance the effect of MAFLD on the risk of MI and stroke. In conclusion, a diagnosis of MAFLD is associated with an increased risk of MI or stroke, and the assessment of MAFLD and its subtypes should be a component of the cardiovascular risk assessment.

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis.

AIMS: Vascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. METHODS AND RESULTS: The case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17-13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated ß-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE-/- mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. CONCLUSIONS: SAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

Associations of Macronutrients Intake With MRI-determined Hepatic Fat Content, Hepatic Fibroinflammation, and NAFLD.

CONTEXT: A healthy lifestyle is the cornerstone of management in nonalcoholic fatty liver disease (NAFLD). However, the associations between dietary macronutrient composition and different aspects of NAFLD pathology are unclear and dietary recommendations for NAFLD are lacking. OBJECTIVE: This work aimed to evaluate the associations of dietary macronutrient composition with hepatic steatosis, hepatic fibroinflammation, and NAFLD. METHODS: In this cross-sectional study, a total of 12 620 UK Biobank participants who completed both the dietary questionnaire and magnetic resonance imaging (MRI) examination were included in this study. Dietary consumption of macronutrient was self-reported and calculated. MRI-determined hepatic fat content, fibroinflammation, and NAFLD were estimated. RESULTS: First, we found that saturated fatty acid (SFA) intake was associated with higher hepatic steatosis, fibroinflammation, and NAFLD prevalence. In contrast, higher fiber or protein intake was reversely correlated with hepatic steatosis and fibroinflammation. Interestingly, starch or sugar intake was significantly associated with hepatic fibroinflammation, whereas monounsaturated fatty acid (MUFA) intake was negatively correlated with hepatic fibroinflammation. Isocaloric analysis revealed that replacing SFA with sugar, fiber, or protein was significantly associated with a reduction in hepatic steatosis, while replacing starch, sugar, or SFA with protein or MUFA was significantly correlated with a decrease in hepatic fibroinflammation. CONCLUSION: Overall, our results demonstrate that specific macronutrients are associated with different aspects of NAFLD, and specific dietary compositions should be recommended for distinct NAFLD-risk populations.

Association of MAFLD with end-stage kidney disease: a prospective study of 337,783 UK Biobank participants.

INTRODUCTION: Metabolic dysfunction-associated fatty liver (MAFLD) has been found to be associated with the prevalence of chronic kidney disease (CKD). However, it is unknown whether MAFLD is associated with CKD development and the incidence of end-stage kidney disease (ESKD). We aimed to clarify the association between MAFLD and incident ESKD in the prospective UK Biobank cohort. METHODS: We analyzed the data of 337,783 UK Biobank participants and relative risks for the ESKD were calculated by using the Cox regression analysis. RESULTS: Among 337,783 participants over a median duration of 12.8 years follow-up, a total of 618 ESKD cases were diagnosed. Participants with MAFLD were twice likely to develop ESKD (hazard ratio [HR] 2.03, 95% confidence interval [CI] 1.68-2.46, p < 0.001). The association of MAFLD with ESKD risk remained significant in both non-CKD and CKD participants. Our results also showed that there were graded associations between liver fibrosis scores and the risk of ESKD in MAFLD cases. Compared to non-MAFLD individuals, the adjusted HRs for incident ESKD in MAFLD patients with increasing levels of NAFLD fibrosis score were 1.23 (95% CI 0.96-1.58), 2.45 (1.98-3.03) and 7.67 (5.48-10.73), respectively. Furthermore, the risking alleles of PNPLA3 rs738409, TM6SF2 rs58542926, GCKR rs1260326 and MBOAT7 rs641738 amplified the MAFLD effect on ESKD risk. In conclusion, MAFLD is associated with incident ESKD. CONCLUSION: MAFLD may help identify the subjects at high risk of ESKD development and MAFLD interventions should be encouraged to slow down CKD progression.

Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and Its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease.

BACKGROUND: Accumulating evidence has shown that disorders in the gut microbiota and derived metabolites affect the development of atherosclerotic cardiovascular disease (ASCVD). However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of their alterations remain unclear. METHODS: We performed integrated microbiome-metabolome analysis of 30 patients with coronary artery disease (CAD) and 30 age- and sex-matched healthy controls to identify CAD-associated microbial metabolites, which were then assessed in an independent population of patients with ASCVD and controls (n=256). We further investigate the effect of CAD-associated microbial metabolites on atherosclerosis and the mechanisms of the action. RESULTS: Indole-3-propionic acid (IPA), a solely microbially derived tryptophan metabolite, was the most downregulated metabolite in patients with CAD. Circulating IPA was then shown in an independent population to be associated with risk of prevalent ASCVD and correlated with the ASCVD severity. Dietary IPA supplementation alleviates atherosclerotic plaque development in ApoE-/- mice. In murine- and human-derived macrophages, administration of IPA promoted cholesterol efflux from macrophages to ApoA-I through an undescribed miR-142-5p/ABCA1 (ATP-binding cassette transporter A1) signaling pathway. Further in vivo studies demonstrated that IPA facilitates macrophage reverse cholesterol transport, correlating with the regulation of miR-142-5p/ABCA1 pathway, whereas reduced IPA production contributed to the aberrant overexpression of miR-142-5p in macrophages and accelerated the progression of atherosclerosis. Moreover, the miR-142-5p/ABCA1/reverse cholesterol transport axis in macrophages were dysregulated in patients with CAD, and correlated with the changes in circulating IPA levels. CONCLUSIONS: Our study identify a previously unknown link between specific gut microbiota-derived tryptophan metabolite and ASCVD. The microbial metabolite IPA/miR-142-5p/ABCA1 pathway may represent a promising therapeutic target for ASCVD.

Supplementation with Sodium Butyrate Modulates the Composition of the Gut Microbiota and Ameliorates High-Fat Diet-Induced Obesity in Mice.

BACKGROUND: Short-chain fatty acids (SCFAs) have been reported to ameliorate obesity. However, the underlying mechanisms require further investigation. OBJECTIVE: The aim of this study was to determine the role of butyrate, an SCFA, in the regulation of obesity, low-grade chronic inflammation, and alterations of microbiota composition in mice. METHODS: Male C57BL/6J mice, 4-5 wk of age, were divided into 3 groups (n = 8 mice/group): low-fat diet (LFD; 10% energy from fat), high-fat diet (HFD; 45% energy from fat), or high-fat diet plus sodium butyrate (HSB). HSB mice received sodium butyrate at a concentration of 0.1 M in drinking water for 12 wk. Measures of inflammation, obesity, and intestinal integrity were assessed. Serum lipopolysaccharide (LPS) concentrations were measured in the 3 groups. Fecal samples were collected for gut microbiota analysis. RESULTS: In HFD mice, body weight gain and hepatic triglyceride (TG), serum interleukin-6 (IL-6), and serum tumor necrosis factor (TNF)-α levels were 1-4 times higher than those in LFD mice (P < 0.05); they were 34-42% lower in HSB mice compared with HFD mice (P < 0.05). The HFD group had 28%-48% lower mRNA expression of both Tjp1 and Ocln in the ileum and colon compared with levels in LFD or HSB mice (P < 0.05), whereas there was no difference in expression levels between LFD and HSB mice. Furthermore, in HSB mice, serum LPS concentration was 53% lower compared with that in HFD mice but still 23% higher than that in LFD mice (P < 0.05). Results from principal component analysis showed that HSB and LFD mice had a similar gut microbiota structure, which was significantly different from that in HFD mice (P < 0.05). CONCLUSIONS: Sodium butyrate administration beneficially changed HFD-induced gut microbiota composition and improved intestinal barrier, leading to lower serum LPS concentrations. These changes may correspond with improvements in obesity-related lipid accumulation and low-grade chronic inflammation.

Adropin protects against liver injury in nonalcoholic steatohepatitis via the Nrf2 mediated antioxidant capacity.

Adropin, a secretory signal peptide, has shown beneficial effects on improving glucose homeostasis and dyslipidemia. However, whether this peptide affects nonalcoholic steatohepatitis (NASH) has remained unclear. In this study, the serum adropin levels, liver injury and oxidative stress were measured in diet-induced NASH mice. Adropin knock-out mice and palmitate treated primary hepatic cells were used to investigate the influence of adropin on liver injury. Our results show that serum adropin levels were decreased and negatively correlated with liver injury in NASH mice. Knockout of adropin significantly exacerbated hepatic steatosis, inflammatory responses and fibrosis in mice after either methionine-choline deficient diet (MCD) or western diet (WD) feeding. And the treatment with adropin bioactive peptides ameliorated NASH progression in mice. Adropin alleviated hepatocyte injury by upregulating the expression of Gclc, Gclm, and Gpx1 in a manner dependent on Nrf2 transcriptional activity and by increasing the glutathione (GSH) levels. And adropin significantly increased CBP expression and promoted its binding with Nrf2, which enhanced Nrf2 transcriptional activity. Furthermore, AAV8-mediated overexpression of hepatic Nrf2 expression functionally restored the liver injury induced by adropin-deficiency MCD-fed mice. These findings provide evidence that adropin activates Nrf2 signaling and plays a protective role in liver injury of NASH and therefore might represent a novel target for the prevention and treatment of NASH.

Coenzyme Q10 attenuates high-fat diet-induced non-alcoholic fatty liver disease through activation of the AMPK pathway.

Coenzyme Q10 (CoQ10) is a well-known anti-adipogenic factor that possesses the capability to regulate non-alcoholic fatty liver disease (NAFLD). However, the mechanism by which CoQ10 acts on NAFLD is still unclear. In this study, the role of CoQ10 in the prevention of NAFLD was investigated in vivo and in vitro. C57BL/6J mice were fed a normal diet, high-fat diet (HFD) or HFD supplemented with CoQ10 (1800 mg kg-1 HFD) for 24 weeks. HepG2 cells were treated with sodium palmitate for investigating the mechanism of action of CoQ10 on NAFLD. The results showed that CoQ10 alleviated HFD-induced weight gain and NAFLD, accompanied by an anti-hyperlipidaemia effect, by reducing the serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels. Importantly, CoQ10 could downregulate the expression of sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS), which are related to lipid synthesis, and upregulate the expression of peroxisome proliferator-activated receptors α (PPARα) and carnitine palmitoyltransferase-1 (CPT-1) associated with fatty acid oxidation. Similar to the results from mice, treatment with CoQ10 alleviated sodium palmitate-induced hepatocyte steatosis via the inhibition of lipogenesis and promotion of fatty acid oxidation. However, Compound C, as an AMPK inhibitor, could significantly block the benefits derived from CoQ10 treatment. In conclusion, CoQ10 could serve as an AMPK activator and regulate the hepatic lipid metabolism to inhibit the abnormal accumulation of hepatic lipids and prevent NAFLD progression.

Cigarette smoking as a risk factor for type 2 diabetes in women compared with men: a systematic review and meta-analysis of prospective cohort studies.

BACKGROUND: Few studies have investigated the effect of smoking on type 2 diabetes in women compared with men, even though several epidemiological studies provided a clear picture of the risk among the entire population. METHODS: We systematically searched PubMed and Embase up to August 2017 for prospective studies that were stratified by sex with measures of the relative risk (RR) for type 2 diabetes and current smoking compared with non-smoking. The sex-specific RRs and their ratios (RRRs), comparing women with man, were pooled using random-effects models. RESULTS: Seventeen articles were identified including 20 prospective cohorts with 5 077 289 participants and 223 084 incident cases of type 2 diabetes. The pooled RRR suggested a similar risk of type 2 diabetes associated with smoking in women compared with men (RRR: 0.98, 95% confidence interval [CI]: 0.96-1.01). Furthermore, no significant sex difference in the RR was found between former smokers and those who had never smoked (RRR: 0.98, 95% CI: 0.92-1.04). CONCLUSIONS: The findings of this meta-analysis indicate that female smokers had similar risk of type 2 diabetes with male smokers.

Association of glycated hemoglobin with the risk of advanced fibrosis in non-alcoholic fatty liver disease patients without diabetes.

BACKGROUND: Association of diabetes with non-alcoholic steatohepatitis has been well documented. However, it remains unclear whether there is an association between levels of glycated hemoglobin (HbA1c) with severity of non-alcoholic fatty liver disease (NAFLD). This study was aimed to explore the relationship between levels of HbA1c and the risk of advanced fibrosis in patients with NAFLD. METHODS: A cross-sectional study was performed on 4826 apparently healthy Chinese, who underwent a health check between January 2015 and December 2016. NAFLD was defined as hepatic steatosis on ultrasonography in the absence of excessive alcohol use or other identifiable causes. The risk of advanced fibrosis was assessed by NAFLD fibrosis Score. RESULTS: Among 4826 individuals studied, 1630 were diagnosed with NAFLD. In a multivariable-adjusted model, high HbA1c levels were associated independently with increased prevalence of NAFLD. The adjusted odds ratio [95% confidence interval (95% CI)] for NAFLD, when compared with the highest HbA1c quartile and the lowest HbA1c quartile, was 2.72 (2.07-3.58; P for trend < 0.001). A strong association was also observed between HbA1c level and the risk of fibrosis in patients with NAFLD in multivariable analyses, with the extreme-quartile odds ratio of 2.69 (95% CI: 1.60-4.53; P for trend < 0.001). This association remained significant even in subjects without diabetes. CONCLUSIONS: We concluded that high HbA1c level was associated strongly and independently with increased risk of advanced fibrosis in NAFLD patients without diabetes.

Serum Bioavailable and Free 25-Hydroxyvitamin D Levels, but Not Its Total Level, Are Associated With the Risk of Mortality in Patients With Coronary Artery Disease.

RATIONALE: Bioavailable and free 25-hydroxyvitamin D (25(OH)D) are emerging measurements of vitamin D. Whether serum bioavailable or free 25(OH)D level is associated with mortality in patients with coronary artery disease (CAD) is unknown. OBJECTIVE: Our aim is to determine the potential association between serum total, bioavailable, and free 25(OH)D levels and the risk of mortality among patients with CAD. METHODS AND RESULTS: We measured serum 25(OH) levels in 1387 patients with angiographically confirmed CAD from the Guangdong Coronary Artery Disease Cohort. Serum DBP (vitamin D-binding protein) levels were measured using a polyclonal immunoassay, and serum-free 25(OH)D levels were measured using a 2-step immunoassay. Bioavailable 25(OH)D levels were calculated using a previously validated formula. By the median follow-up time of 6.7 years, 205 patients had died, including 134 deaths from cardiovascular diseases. In multivariate analyses, low serum bioavailable 25(OH)D level was significantly associated with increased risks of mortality, independent of established cardiovascular risk factors, features and treatments of CAD, factors associated with vitamin D and mineral metabolism, and CRP (C-reactive protein). The multivariable-adjusted hazard ratios across quartiles of bioavailable 25(OH)D were 1.79, 1.35, 1.36, and 1.00 for all-cause mortality ( P for trend=0.01) and 2.58, 1.85, 1.73, and 1.00 for cardiovascular mortality ( P for trend=0.001), respectively. Serum-free 25(OH)D level was inversely associated with the risk of mortality, with the extreme-quartile hazard ratios of 1.64 for all-cause mortality ( P for trend=0.024) and 1.97 for cardiovascular mortality ( P for trend=0.013). In contrast, serum total 25(OH)D level was not significantly associated with all-cause mortality or cardiovascular mortality. CONCLUSIONS: Lower serum bioavailable and free 25(OH)D levels rather than total 25(OH)D level are independently associated with an increased risk of all-cause mortality and cardiovascular mortality in a population-based CAD cohort.

Urban-Rural Differences in Cardiovascular Disease Risk Factors: A Cross-Sectional Study of Schoolchildren in Wuhan, China.

BACKGROUND: China's rapid population growth and urban migration has developed healthcare inequity across the urban-rural divide. Past studies comparing cardiovascular disease (CVD) risk factor prevalence amongst urban-rural Chinese children are sparse and conflicting. We examined the association between urban-rural residence and risk of offspring CVD in Chinese children. METHODS: A cross-sectional study was conducted in Wuhan, China, during May and June 2010. CVD risk factors include; waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), triglycerides (TG), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, body mass index (BMI), cardiorespiratory fitness (CRF), metabolic syndrome (MetS), and metabolic risk score (MRS). Analysis of covariance and multivariable logistic regression were used to estimate associations between urban-rural residence and offspring CVD risks. FINDINGS: A total of 579 Chinese children (338 boys and 241 girls) aged 9.6 (0.7) years participated in this study. Rural boys had significantly lower CRF and higher FBG, TG, and MRS, while urban boys had significantly higher LDL and DBP. Rural girls had significantly higher BMI, FBG, and TG, as well as lower CRF. Rural children were at increased risks for decreased CRF, elevated MRS, and TG, (OR:2.04, 95%CI:1.29-3.25), (OR:2.33, 95%CI:1.50-3.62), and (OR:2.40, 95%CI:1.62-3.57), respectively. Rural girls and mothers were at increased risks for overweight(OR:7.19, 95%CI:1.64-31.6)/obesity (OR:1.683, 95%CI:1.01-2.82). However, rural boys and fathers were less likely to have overweight(OR:0.62, 95%CI:0.34-1.12)/obesity (OR:0.68, 95%CI:0.48-0.97). CONCLUSIONS: Rural residence was significantly associated with increased CVD risks amongst Chinese children. It is important to provide interventions aiming at China's urban-rural healthcare inequity and community-based approaches that reduce familial CVD risk.